Susceptibility to Heavy Metals and Characterization of Heterotrophic ...

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bacteria could intervene in detoxifying the heavy-metal-rich microenvironment of the worm (2, 39). In a preliminary study, heterotrophic bacteria colonizing.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3308-3314 0099-2240/90/113308-07$02.00/0 Copyright © 1990, American Society for Microbiology

Vol. 56, No. 11

Susceptibility to Heavy Metals and Characterization of Heterotrophic Bacteria Isolated from Two Hydrothermal Vent Polychaete Annelids, Alvinella pompejana and Alvinella caudata CHRISTIAN JEANTHON* AND DANIEL PRIEUR

Centre National de la Recherche Scientifique, LP 4601, Station Biologique, B.P. 74, 29682 Roscoff Cedex, France Received 10 May 1990/Accepted 23 August 1990

Specimens of alvinellid polychaetes and their tubes were collected in the Parigo hydrothermal vent field on the East Pacific Rise (13°N) in October and November 1987. Heterotrophic bacterial strains were isolated on metal-amended media from the tube and dorsal integument of one specimen of Alvinella pompejana, from the dorsal integument of another from the whole integument of a specimen of Alvinella caudata, and from undetermined alvinellid tubes. The strains were characterized and tested for susceptibility to five heavy metals by using a microdilution method for MIC determinations. All strains were gram-negative rods. Most of them were characterized by the ability to degrade Tween 80 and gelatin and to produce hydrogen sulfide from cysteine. Numerous strains, from all sample origins, displayed resistance to cadmium, zinc, arsenate, and silver and tolerated high amounts of copper. Metal resistance was exhibited by 92.3% of the total isolates. The occurrence of multiply resistant bacteria may demonstrate an adaptation of alvinellid-associated microflora to the general enrichment of metals in the hydrothermal vent environment. Some non-vent epibiotic bacterial associations have been described and/or studied on the marine priapulid worm Halicryptus spinulosus (35), on the copepod Acartia tonsa (46), and on the crab Cancer irroratus (8). However, little is known about the functional role of epibioses. Epibacteria associated with H. spinulosus might be able to detoxify hydrogen sulfide (35). In the case of bacteria-Alvinella associations, a trophic relationship has been suggested but not clearly demonstrated (2). The hydrothermal fluids are particularly rich in heavy metals (11), and the contamination of vent invertebrate soft tissues has been reported (21, 42). It has been suggested that these epibiotic bacteria could intervene in detoxifying the heavy-metal-rich microenvironment of the worm (2, 39). In a preliminary study, heterotrophic bacteria colonizing the dorsal integument of A. pompejana and its tube (39) were reported to be resistant to high concentrations of heavy metals (C. Jeanthon and D. Prieur, Prog. Oceanogr., in press). The objective of the present study was to characterize a large number of alvinellid-associated bacteria isolated on metal-amended media and to assess their susceptibility to heavy metals.

Active deep-sea hydrothermal vents discovered on the East Pacific Rise are all characterized by biological communities consisting of a variety of specially adapted animals. The spectacular tube-worm Riftia pachyptila and the large mussel Bathymodiolus thermophilus colonize vent sites from 13°N next to the zones where anoxic vent water, rich in hydrogen sulfide, mixes with surrounding oxygenated seawater (22). The harboring by these animals of chemosynthetic bacteria led to the discovery of sulfide-exploiting symbioses (9, 12). In contrast, Alvinella pompejana (16) and Alvinella caudata, belonging to the recently described Alvinellidae family (17), do not contain intracellular bacteria. Both worms build mineralized organic tubes on the walls of sulfide diffusers in areas of active mixing of hot, acidic, metal-rich fluid with cold, well-oxygenated seawater (13). Although polychaetous annelids commonly lack associated bacteria (43), both species bear dense epibiotic microflora (14, 20). Rod-shaped, prosthecate, and small spiral-curved bacteria are scattered on the surface of the worm integument, whereas clumplike assemblages are inserted in the intersegmentary spaces (14). Visible autotrophic filamentous bacteria (51) are located on the dorsal expansions of A. pompejana and on the posterior parapodia of A. caudata (14). The inner part of the worm tube is also densely covered with bacterial communities of different morphological types (rod shaped and principally filamentous) (15). Other examples of epibacterial colonization of hydrothermal vent animals have been reported: bacteria were found on the bristles of notopodia and on the elytra of polynoids of the genus Lepidonotopodium collected at 13°N (37), dense colonies of filamentous bacteria were described on the epithelial surface of gills of an undescribed archeogasteropod limpet from vents of the Juan de Fuca Ridge (12), and microbial mats have been observed on the periostracum of thermal vent mussels from the Galapagos vents (27).

*

MATERIALS AND METHODS Polychaete collection and preparation. Both worm species, A. pompejana and A. caudata, and undetermined alvinellid tubes samples were collected in November and December 1987 from the Parigo vent field on the East Pacific Rise (12048'56"N, 103°56'72"W) at a depth of 2,600 m during the Hydronaut cruise. Two specimens of A. pompejana (designated here as A. pompejana 1 and 2), one specimen of A. caudata, the tube of A. pompejana 1, and undetermined alvinellid tubes were collected by using the port manipulator of the submersible Nautile and placed in an insulated box for the trip to the surface. Upon recovery, worms and tubes were rinsed in sterile seawater. The dorsal integument of both specimens of A. pompejana, a whole specimen of A. caudata, the tube of A. pompejana 1, and the tubes from undetermined alvinellids were ground in sterile seawater

Corresponding author. 3308

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VOL. 56, 1990

TABLE 1. Numbers and sources of isolates included in this study Source

A. pompejana 1 A. pompejana 2 A. caudata Tube of A. pompejana 1 Alvinellid tubes

No. of isolates isolated on media amended by: Cadmium Zinc Copper Arsenate Silver Selenate (Cd2+) (Zn2+) (Cu2+) (As043-) (Ag+) (SeO4 2)

6 1 8 5

16 12 18 19

10 17 10 21

25 15 11 9

27 13 7 22

0 0 0 0

5

0

1

12

7

2

with a grinder (Polytron; Kinematica, Lucerne, Switzerland). Isolation of microorganisms. Ground material was diluted, and dilutions were plated onto metal-amended media. Metalamended media were prepared by adding to the medium of Oppenheimer and Zobell (36) either 80 ,ug of Cd ml-', 100 jig of Zn ml-', 90 ,ug of Cu ml-', 1,850 ,ug of As043- ml-1, or 10 ,ug of Ag ml-'. Plates were incubated until returned to the laboratory. Colonies from enumeration plates were randomly picked and streaked onto agar medium for purification. The numbers and the origins of strains carried through the full test series are listed in Table 1. Susceptibility testing. (i) Chemicals. The following five tested metals were purchased from Carlo Erba Inc.: CdCl2 2.5H2O, ZnCl2, CuCl2 2H20, Na2HAsO4-7H20, and AgNO3. Stock solutions were made in distilled water, sterilized by filtration through membrane filters (pore size, 0.22 ,um) (Nuclepore Corp., Pleasanton, Calif.), and stored in sterile flasks in the dark at 4°C for no longer than 1 day. All chemicals were of analytical reagent grade. (ii) Growth conditions. Susceptibility tests were conducted in a liquid culture medium containing 1.25 g of peptone, 0.25 g of yeast extract, 750 ml of seawater, and 250 ml of distilled water. In this medium, organic matter concentrations used in the isolation medium were lowered in order to minimize metal complexation. Metal-amended media were prepared by adding increasing amounts of metal stock solutions to autoclaved medium. The highest metal concentrations tested were 175 ,ug of Cd ml-', 175 ,ug of Zn ml-', 130 ,ug of Cu ml-', 3,700 ,ug of As043- ml-1, and 40 ,ug of Ag ml-'. The pHs were adjusted to 7 except for the copper-containing medium where pH was limited to 5.5 in order to avoid precipitation. Media were poured into 96-well plates and inoculated with mid-logarithmic-phase cultures. (iii) MIC determinations. Criteria for resistance were based on MIC frequency distributions (32). The MIC was defined as the lowest concentration of the metal that inhibited growth after an incubation of 10 days at room temperature in the dark. Growth media without antimicrobial agents and inoculated with test microorganisms were used as controls. Growth of the inocula was measured by optical density at 620 nm with a microwell plate reader (Titertek Multiskan MC; Flow Laboratories S. A., Puteaux, France). The absence of growth was determined by the detection threshold of the plate reader. Characterization of isolates. Each strain was examined for 30 separate characteristics. All test media were inoculated with cultures grown on Marine Broth (Difco) for 1 day at 18°C. Whenever possible, Marine Broth or Marine Agar (Difco) was used as the basal medium for the test media. To assess possible error, tests were performed in triplicate. Strains were observed for morphology and motility (phase-contrast microscopy) and Gram staining.

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(i) Biochemical tests. The oxidase reaction was determined with 1-day cultures by using the procedure of Kovacs (30). H2S production and Tween 80 degradation were recorded after 7 days by using the methods described by Rodina (41) and Sierra (44). Proteolytic activity on gelatin and P-galactosidase activity were tested as described by Kohn (29) and Lanyi (33). The presence of constitutive arginine dihydrolase system, indole production, nitrate and nitrite reduction, and starch hydrolysis were determined in 96-well plates by using previously described methods (5, 33). Ninety-six-well plates were also used for testing oxidative and fermentative utilization of glucose and acid production from the following sterile-filtered carbohydrates (final concentration; 1% [wt/ vol]: L-arabinose, D-ribose, xylose, D-galactose, D-mannose, D-fructose, sucrose, lactose, maltose, starch, mannitol, sorbitol, and glycerol. (ii) Reference strains. The following marine bacterial strains, available in our laboratory, were included in this analysis as references: Deleya marina DSM 50416, Deleya pacifica NCMB 1977, Deleya cupida NCMB 1978, Deleya venusta NCMB 1979, Deleya aesta NCMB 1980, Alcaligenes aquamarinus DSM 30161, Pseudomonas doudoroffii NCMB 1965, Pseudomonas nautica NCMB 1967, Halomonas elongata NCMB 2198, Vibrio harveyi DSM 2332, Vibrio natriegens DSM 759, Vibrio costicola NCMB701, and Vibrio anguillarum NCMB 6. (iii) Computer analysis. The characters were coded 1 for positive or present and 0 for negative or absent. Strain similarities were estimated with the simple matching coefficient SSM (47), and cluster analysis was carried out by using the average linkage method (45). RESULTS Heavy metal resistance. The distribution of the MIC values of each metallic ion for all of the strains examined is shown in Fig. 1. Patterns of resistance of cadmium, zinc, silver, and arsenate showed a clear-cut distinction between the sensitive and the resistant strains. Critical MICs above which strains were considered resistant were 90 ,ug of Cd ml-', 70 ,ug of Zn ml-', 3,500 ,ug of As043- ml-', and 20 ,ug of Ag ml-'. As the distribution pattern of copper resistance revealed only one peak, copper-resistant strains could not be detected. Metal resistance was exhibited by 92.3% of the total isolates. Table 2 summarizes the percentages of isolated strains resistant to each metal. From this table, three observations can be made: first, the small number of cadmium- and silver-resistant isolates from A. pompejana 2; secondly, the high number of arsenate-resistant strains in all samples, most of which were not inhibited by the highest concentration of arsenate tested; and thirdly, the resistance to silver of 51.8% of the isolates from A. pompejana 1. The frequencies of resistance to metals, except silver, were quite similar for strains isolated from the dorsal integument and strains from the tube of A. pompejana 1. Heavy metal multiple resistance. Only a few strains did not display resistance to the metals tested, whereas 57% of the total isolates exhibited resistance to more than one metal. The percentages of strains showing multiple resistance (Table 3) were highest for the A. pompejana 1 isolates. Strains which displayed multiple resistance to metals were recovered from all the samples. Moreover, strains isolated from A. pompejana 1 were resistant to an average of 2.0 different metals, while the

APPL. ENVIRON. MICROBIOL.

JEANTHON AND PRIEUR

3310

la 40

la _@

q._

,,

Coppcr

Zinc

Cadmium

30

7

10 20 40 50 70 90 100 130>130

10 20 40 50 70 90 100130175>175

MIC (pg/mi)

MI I C ( pg/m I)

MIC (pg/mi)

Silvcr

Arscnatc '0

80

I)

60

ac

40

a

-030-

C

o 4-

20

-.0

20

0

0

0

2.0 2.2 2.4 2.6 2.8 3.0 3.1 3.3 3.5 3.7 >3.7

o) 3

FIG. 1. MIC distribution patterns of cadmium, zinc, samples.

copper,

1.7, 1.7, and 1.4. Strain characterization and clustering. The characteristics of all phena obtained in this study are given in Table 4. All of the strains studied were gram-negative rods. Some characteristics were frequently encountered, such as the presence of catalase (98% of the strains) and oxidase (79%), the production of H2S from cysteine (86%), and the degradation of Tween 80 (90%) and gelatin (87%). The SSM coefficient yielded results indicating that 291 strains, i.e., 97% of the total, were recovered in 18 clusters defined at a 75 to 88% similarity level (Fig. 2). Most strains examined in this study (242 out of 299) were clustered in only seven phena (1, 2, 6, 7, 13, 15, 17). Bergey's Manual of Systematic Bacteriology (31), The Prokaryotes (48), and specialist diagnostic keys and tables were consulted for presumptive identification of the most representative phena (phena 1, 2, 6, 7, 13, 15, and 17). The intergroup similarities for the clusters ranged from TABLE 2. Frequency of heavy metal resistance in 299 strains isolated from alvinellid samples Source

No. of strains resistant toa:

Cd2+

Zn2+

7

10

15

20 25 30 40

arsenate, and silver for 299 heterotrophic strains isolated from alvinellid

corresponding values for strains isolated from A. pompejana 2, from A. caudata, from the tube of A. pompejana, and from undetermined alvinellid tubes were, respectively 1.3,

Total no. of strains

s

5

MIC (pg/mi)

MIC (mg/ml)

As043-

Ag+

A. pompejana 1 84 40.0 36.5 70.6 51.8 A. pompejana 2 58 12.1 39.6 69.0 10.3 A. caudata 54 31.5 44.4 79.6 14.8 Tube of A. pom76 43.4 39.5 69.7 17.1 pejana 1 Alvinellid tubes 27 33.3 14.8 77.8 18.5 " We could not detect Cu-resistant strains since the distribution pattern of Cu resistance shows only one peak.

52 to 88%, suggesting a marked diversity among the taxa recovered from the alvinellid samples. Cluster 1 formed at 82%. All strains were oxidase positive and degraded gelatin and Tween 80. Starch hydrolysis and P-galactosidase activity were shown by a large majority of the strains. Of the strains, 53% were isolated from A. pompejana 1 and 53% were isolated on silver agar plates. Cadmium and silver resistance were displayed by 41.8 and 54.5%, respectively, of the strains, and 31% of all strains exhibited resistance to both metals. In this cluster, six strains, all isolated from A. pompejana 1, were resistant to cadmium, zinc, arsenate, and silver. Cluster 2, which joined at 84%, is composed entirely of oxidase- and catalase-positive strains with lipase activity. Forty-three percent were isolated from alvinellid tubes, representing more than half of the alvinellid tube isolates. Cluster 6, formed at 89%, consisted of oxidase-positive strains which did not produce acid from 10 of the carbohydrates tested. Most strains were isolated on silver agar plates and from the tube of A. pompejana 1. One strain demonstrated resistance to cadmium, zinc, arsenate, and silver. TABLE 3. Percentages of heavy-metal-multiresistant strains isolated from different alvinellid samples % of isolates resistant to indicated

no. of metals

Source 0

1

2

3

4

A. pompejana 1 A. pompejana 2 A. caudata Tube of A. pompejana 1

2.3 19.0 7.4 7.9

27.0 37.9 37.0 31.6

47.1 37.9 38.9 44.7

16.5 3.4 11.1 14.5

7.1 1.7 5.6 1.3

Alvinellid tubes

7.4

55.6

22.2

14.8

0

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TABLE 4. Characteristics of clusters 1 to 18 % of positive responses in cluster no.:

Characteristics

No. of strains Source A. pompejana 1 A. pompejana 2 A. caudata Tube of A. pompejana 1 Alvinellid tubes Isolation medium Cadmium Zinc

Copper Arsenate Silver Selenate

1

2

6

7

10

11

12

13

14

16

17

18

55

33

3

3

3

6

33

36

3

2

11

2

4

17

2

26

4

42

9

53 24 13 5

21 30 6 0

66 0 0 0

0 0 0 66

0 33 50 0

15 3 12 70

19 45 36 0

33 33 33 0

0 0 50 50

9 27 18 37

50 50 0 0

25 25 25 0

29 18 47 6

0 0 0 100

23 4 4 69

33 5 19 43

5

43

33

33

17

0

0

0

0

9

0

25

0

0

0

0 75 25 0 0

0

11 0 11 23 45

9 13 3 22 53 0

0 9 9 46 36 0

33 0 0 33 33 0

0 33 33 0 33 0

17 0 17 66 0 0

0 15 6 12 67 0

0 47 30 17 6 0

33 0 0 66 0 0

0 0 50 0 50 0

9 36 9 46 0 0

0 50 0 50 0 0

0 0 25 25 50 0

35 35 18 6 6 0

50 0 0 0 50 0

4 15 58 23 0 0

25 25 50 0 0 0

14 26 31 24 5 0

0 3 11 33 0 23

100 82 100 4

100 85 100 3

100 0 100 33

100 66 100 33

100 100 100 100

100 91 100 3

100 97 100 94

0 1 100 100 100 100 100 91 50 100 100 100 100 33 0 55 100

100 75 100 25

100 82 100 100

100 100 100 100

100 58 100 100

100 75 100 100

100 95 100 62

100 89 100 11

0 98 100 2 0 2 0 98 94

0 100 100 6 0 3 0 97 24

0 100 100 0 0 0 0 100 66

0 100 33 0

0 0 0 0 66

0 100 100 33 0 33 0 50 33

0 91 100 82 33 0 0 91 0

0 100 25 94 0 0 0 100 11

0 100 100 100 0 0 0 100 0

0 50 100 100 100 0 0 50 0

0 91 82 64 100 0 0 100 0

0 100 0 0 0 0 0 100 0

0 100 100 0 0 50 0 100 75

0 100 100 6 0 88 0 0 0

0 100 100 0 0 0 0 0 0

0 100 88 8 0 4 11 88 4

0 100 100 25 0 100 0 0 0

106 98 98 100 0 100 86 98 88

100 100 100 100 0 0 100 89 11

100 91 100

97 3 100

100 100 100

66 0 100

17 0 33

97 0 97

97 3 100

100 0 66

50 50 100

64 0 91

100 0 100

100 0 100

12 0 12

0 0 0

92 4 100

0 0 0

98 98 98

89 100 89

2 7 5 0 53 2 4 16 0 98 2 4 0 0

36 0 0 9 6 94 67 82 0 100 3 18 3 0

66 0 0 0 66 33 66 100 33 100 100 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 33

0 0 0 17 0 0 0 0 0 17 0 0 0 0

0 21 0

0 0 0

0 0 0 3 0 0 3 0 0 0 39

0 3 3 3 0 0 6 3 3 0 3

0 0 0 0 66 0 100 0 0 33 0 0 0 0

0 100 0 0 0 0 0 0 0 50 50 0 0 0

55 64 9 9 91 64 91 100 0 27 50 64 18 45

0 0 50 50

75 75 75 100

0 100 100 100 0 100 9 0 0 100

100 50 0 0 0 50 50 25 0 25

100 100 100 100 100 100 100 100 65 94 100 59 100 94

50 100 100 0 100 50 100 100 50 100 0 100 100 0

96 100 100 100 100 100 100 100 88 96 88 96 85 88

100 0 75 100 75 100 50 75 0 75 0 0 0 0

90 98 0 100 100 57 100 100 0 100 95 100 2 100

0 100 0 100 0 0 89 100 0 100 100 100 11 100

4

5

8

9

15

Morphology Rods

Motility Gram negative Growth without NaCl Biochemical tests Fementative metabolism Catalase Oxidase Nitrate reduction Nitrite reduction Arginine dihydrolase Indole H2S from cysteine ONPGa Degradation tests Gelatin Starch Tween 80 Acid from: L-Arabinose D-(-)-Ribose D-Xylose D-Glucose D-Galactose D-(+)-Mannose D-Fructose Sucrose Lactose D-(+)-Maltose Starch D-Mannitol D-Sorbitol

Glycerol

a ONPG, o-Nitrophenyl-,-D-galactopyranoside.

Cluster 7 joined at 89% and contained catalase-positive strains which all produced H2S from cysteine and degraded Tween 80. All strains were isolated from alvinellid epidermis, and all but two were resistant to zinc. None was resistant to silver, and two of the isolates showed resistance to cadmium. Clusters 1, 2, 6, and 7 with intergroup similarity of 77% were identified as Alteromonas spp. from the production of extracellular lipase, gelatinase, and amylase (cluster 1 only). All strains of clusters 9 and 10 were denitrifying bacteria. Some strains clustered in phenon 10 were not able to reduce nitrate to nitrite. Cluster 13, which joined at 88%, was composed of oxidase- and catalase-positive strains producing acid from 10 of the carbohydrates tested. Few strains were gelatin and

Tween 80 decomposers. All strains but one were resistant to zinc. Three strains isolated on arsenate agar plates were resistant to cadmium, zinc, arsenate, and silver. Members of this cluster were identified as Pseudomonas spp. on the basis of positive responses to oxidase and arginine dihydrolase tests. Strains able to produce fluorescent pigmentation (Pseudomonas fluorescens) are clustered in this phenon. Cluster 15, formed at 83%, consisted of strains degrading Tween 80 and gelatin and producing acid from six of the carbohydrates tested. A high proportion of the strains was isolated from the tube of A. pompejana 1, on copper agar plates, or both. Resistance to zinc was frequently displayed (20 out of 26), but no strain showed silver resistance. On the basis of biochemical features and since the reference strain

APPL. ENVIRON. MICROBIOL.

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PHENON

1

2 3 4

NO. OF STRAINS 55

6

33 3 3 6 33

5

7

36

8

3

9

2

10 11

11 2

12

4

13

17

14

1S

% SIMILARITY

2 26

16

4

17

42

18

9

100

90

80

70

60

50

FIG. 2. Simplified dendrogram prepared by using the Sokal and Michener coefficient (47) and average linkage method of clustering.

P. doudoroffii clustered in this group, members of cluster 15 were assumed to be presumptively Pseudomonas spp. Clusters 17 and 18 were composed entirely of fermentative strains. All strains but one were oxidase positive. Reference strains belonging to the genus Vibrio were recovered in both clusters for which all strains were presumptively assigned to Vibrionaceae. Most isolates were resistant to cadmium (80%), only three of them showed resistance to silver, and eight showed resistance to zinc. Arsenate-resistant strains (72.2% of the total isolates) appeared in all major clusters in high proportions, except in phenon 15. Reference strains other than those cited were not recovered in any cluster determined in this study. DISCUSSION The chemical and physical features of hydrothermal fluids and the dense animal populations in the immediate vicinity of the vents led microbiologists to study the primary production of organic carbon by sulfur-oxidizing bacteria (26) and to isolate thermophilic organisms (28). Bacterial heterotrophic activities were measured in vent-surrounding seawater but were essentially attributed to mixotrophic sulfur-oxidizing bacteria (52). Various hypotheses about the food source for the polychaete worms living closest to the hot water extruding from the vents have been proposed. The possible food sources include bacteria from the rain of black smoker precipitates

(4) and metabolites produced by the abundant communities of filamentous bacteria associated with the dorsal integument of worms or the inner surfaces of their tubes (14). The morphological diversity of worm epibacteria has been described (20), and the metabolic diversity of alvinellidassociated microflora has been established (S. Prieur, S. Chamroux, P. Durand, G. Erauso, P. Fera, C. Jeanthon, L. Leborgne, G. Mevel, and P. Vincent, Mar. Biol., in press). A preliminary study of heterotrophic and mixotrophic bacteria isolated from A. pompejana reported a lack of fermentative metabolism and few proteolytic and amylolytic activities (39). The ability to decompose fatty substrates and to produce H2S from cysteine were the main biochemical characteristics of these isolates. The present study presents the biochemical features of a larger collection of strains isolated from Alvinellidae. The data obtained here show that few fermentative strains are associated with alvinellid epibiotic microflora (51 of 299 strains). The degradation of Tween 80 and the production of H2S from cysteine appear to be very common features, as previously reported (39). Strains demonstrate widespread proteolytic activity. The presence of such microorganisms, promoting the decomposition of organic matter, is known to be ecologically significant in areas of high nutrient concentrations (38). Characterization tests were conducted at a temperature close to the average in situ conditions but, because of technical reasons such as the limited space in pressure vessels, high-pressure conditions which would permit a more complete simulation of the habitat of the isolate could not be provided. A recent study of nonhydrothermal deepsea bacteria showed a change in the enzyme profile when tested at 1 atm (101.29 kPa) as compared with results obtained under in situ pressure (49). A very low rate of degradation activity was measured for strains of similar origin cultivated under in situ conditions (25). Considering the exceptional conditions that exist in hydrothermal environments (unusually high nutrient concentrations and temperatures at such depths), the degradative abilities of the isolates under in situ pressure should provide an interesting field for future research efforts. The presence of large amounts of heavy metals is a major feature of the hydrothermal vent environment, but relatively little is known about the relationships between the metals and the organisms living at these sites. It was not possible to select metals to be studied by using chemical data concerning the 13°N site because such data were scarce an incomplete. However, for another hydrothermal vent site of the East Pacific Rise, the 21°N site, where the faunistic composition of populations was comparable to the one we studied, heavy metal concentrations were measured in vent fluids (18) and in animal tissues and their associated bacteria (21, 42). Arsenic, zinc, and copper were detected in A. pompejana epidermal cells as well as in the associated bacteria and their matrices (21). Arsenic and zinc appeared to be the most abundant elements. In contrast to values in Mytilus edulis from an uncontamined coastal environment, silver exhibited one of the highest enrichments in the hydrothermal vent clam Calyptogena magnifica (42). Heavy metal concentrations measured in undiluted spring fluid were for all metals consistently higher than in the surrounding nonhydrothermal deep-sea water. Zinc and copper, however, were found in amounts about a thousand times higher than those of arsenic, cadmium, and silver (18). Concentrations used to isolate the strains were determined on the basis of a preliminary study (Jeanthon and Prieur,

VOL. 56, 1990

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Prog. Oceanogr.,

in press). Isolation media contained high metal and nutrient concentrations. As there is overwhelming evidence of organic component metal-complexing ability (7, 40), metallic-ion toxicity was consistently reduced. For this reason, the metal susceptibility test conducted here was carried out in liquid medium with low peptone and yeast extract contents. Nevertheless, metal resistance was exhibited by 92.3% of the isolates. The presence of bacteria that are resistant to zinc and/or which tolerate high amounts of copper may be linked to the abundance of these elements in their environment. The percentages of bacteria resistant to arsenate and silver and the level of resistance of the bacteria to both metals reflect the relative toxicity of the metals. Arsenate ions are well known to be one of the less toxic compounds, whereas silver, formerly used as an antimicrobial agent, generally shows a high activity against bacteria. In the literature, great differences exist in the concentrations used to define metal resistance in bacteria (50). The level of bacterial resistance to cadmium, zinc, and arsenate determined here is among the highest reported at this time. As for the biochemical characterization of the strains, the influence of pressure on metal susceptibility was not assayed. It has been reported that this parameter could act in different ways on the effect of heavy metals on the growth of deep-sea isolates (3). Pressure could be ineffective, enhance toxicity, increase growth yield, or protect against a growth inhibitory effect observed at lower pressure. An earlier study (Jeanthon and Prieur, Prog. Oceanogr., in press) reported that the number of bacteria growing on metal-amended media can be in the same order of magnitude as heterotrophic bacteria culturable on the same medium without metal. The metal-amending isolation medium is not the sole metal to which the isolated strain is resistant. Multiresistant bacteria occurred in all samples also, thus demonstrating an adaptation of alvinellid-associated microflora to cope with metal stress. Microorganisms are known to employ a large variety of mechanisms for adaptation to the presence of heavy metal ions (19). Among those mechanisms, it has been reported that hydrogen sulfide production may act as a detoxification process through the precipitation of metal sulfides (1). The high number of H2S-producing isolates recovered in this study may indicate that this mechanism is involved. Resistance to cadmium, zinc, copper, arsenate, and silver can be governed by plasmid-encoded mechanisms (10, 23, 24, 34). Bacteria isolated from the gut of deep-sea amphipods have been found to harbor plasmids, and the occurrence of in situ interspecies transmission has been suggested (53). The screening of plasmid content in alvinellid-associated microflora is now in progress in our laboratory. ACKNOWLEDGMENTS We thank A. M. Alayse-Danet, Chief Scientist of the Hydronaut cruise organized by IFREMER, for inviting us aboard the N.O. Nadir, N. Benbouzid-Rollet for the critical reading of this manuscript, and C. Leroux and J. L. Douville for their technical assistance in computer analysis. Financial support of this research was supplied by grants from Centre National Recherche Scientifique (GDR "Ecoprophyce"), from Programme National pour l'Etude Groupe de Recherche de l'Hydrothermalisme Ocdanique, and from IFREMER (grant no.

882470219). -

1.

LITERATURE CITED

Aiking, H., A. Stijnman, C. van Garderen, H. van Heerikhuizen,

and J. van't Riet. 1984. Inorganic phosphate accumulation and cadmium detoxification in Klebsiella aerogenes NCTC 418

2. 3.

4.

5.

6.

7.

8.

9.

10. 11.

12.

13.

14.

15.

3313

growing in continuous culture. Appl. Environ. Microbiol. 47: 374-377. Alayse-Danet, A. M., F. Gaill, and D. Desbruyeres. 1986. In situ carbonate uptake by bacteria-Alvinella associations. Mar. Ecol. 7:233-240. Arcuri, E. J., and H. L. Ehrlich. 1977. Influence of hydrostatic pressure on the effects of the heavy metal cations of manganese, copper, cobalt, and nickel on the growth of three deep-sea bacterial isolates. Appl. Environ. Microbiol. 33:282-288. Baross, J. A., and J. W. Deming. 1985. The role of bacteria in the ecology of black smoker environments. Biol. Soc. Wash. Bull. 6:355-371. Baumann, P., and L. Baumann. 1981. The marine gram-negative eubacteria: genera Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes, p. 1302-1331. In M. P. Starr, H. Stolp, H. G. Truper, A. Ballows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag KG, Berlin. Belkin, S., D. C. Nelson, and H. W. Jannasch. 1986. Symbiotic assimilation of CO2 in two hydrothermal vent animals, the mussel Bathymodiolus thermophilus and the tube worm Riftia pachyptila. Biol. Bull. 170:110-121. Bird, N. P., J. G. Chambers, R. W. Leech, and D. Cummins. 1985-. A note on the use of metal species in microbiological tests involving growth media. J. Appl. Bacteriol. 59:353-355. Bodamer, J. E., and T. K. Sawyer. 1981. Aufwuchs protozoa and bacteria on the gills of the rock crab, Cancer irroratus Say: a survey by light and electron microscopy. J. Protozool. 28:3546. Cavanaugh, C. M., S. L. Gardiner, M. L. Jones, H. W. Jannasch, and J. B. Waterbury. 1981. Prokaryotic cells in the hydrothermal vent tube worm Riftia pachyptila Jones: possible chemoautotrophic symbionts. Science 213:340-341. Cooksey, D. A. 1987. Characterization of a copper resistance plasmid conserved in copper-resistant strains of Pseudomonas syringae pv. tomato. Appl. Environ. Microbiol. 53:454-456. Corliss, J. B., J. Dymond, L. I. Gordon, J. M. Edmond, R. P. von Herzen, R. D. Ballard, K. Green D. Williams, A. Bainbridge, K. Crane, and T. H. van Andel. 1979. Submarine thermal springs on the Galapagos Rift. Science 203:1073-1083. de Burgh, M. E., and C. L. Singla. 1984. Bacterial colonization and endocytosis on the gills of a new limpet species from a hydrothermal vent. Mar. Biol. 84:1-6. DesbruyOres, D., P. Crassous, J. Grassle, A. Khripounoff, D. Reyss, M. Rio, and M. van Praet. 1982. Donnees ecologiques sur un nouveau site d'hydrothermalisme actif de la ride du Pacifique oriental. C.R. Acad. Sci. 295:489-494. DesbruyOres, D., F. Gaill, L. Laubier, and Y. Fouquet. 1985. Polychaetous annelids from hydrothermal vent ecosystems: an ecological overview. Biol. Soc. Wash. Bull. 6:103-116. Desbruyeres, D., F. Gaill, L. Laubier, D. Prieur, and G. H. Rau. 1983. Unusual nutrition of the "Pompeii worm" Alvinella pompejana (polychaetous annelid) from a hydrothermal vent environment: SEM, TEM, '3C and 15N evidence. Mar. Biol.

75:201-205. 16. Desbruyeres, D., and L. Laubier. 1980. Alvinella pompejana gen. sp. nov., Ampharetidae aberrant des sources hydrothermales de la ride Est-Pacifique. Oceanol. Acta 3:267-274. 17. Desbruyeres, D., and L. Laubier. 1986. Les Alvinellidae, une famille nouvelle d'annelides polychetes infeodees aux sources hydrothermales sous-marines: systematique, biologie et ecologie. Can. J. Zool. 64:2227-2245. 18. Edmond, J. M., and K. L. Von Damm. 1985. Chemistry of ridge crest hot springs. Biol. Soc. Wash. Bull. 6:43-47. 19. Gadd, G. M., and A. J. Griffiths. 1978. Microorganisms and heavy metal toxicity. Microb. Ecol. 4:303-317. 20. Gaill, F., D. Desbruyeres, and D. Prieur. 1987. Bacterial communities associated with "Pompeii worms" from the East Pacific Rise hydrothermal vents: SEM, TEM observations. Microb. Ecol. 13:129-139. 21. Gaill, F., S. Halpern, C. Quintana, and D. Desbruyeres. 1984. Presence intracellulaire d'arsenic et de zinc assocides au soufre chez une Polychete des sources hydrothermales. C.R. Acad. Sci. 298:331-335.

3314

JEANTHON AND PRIEUR

22. Grassle, J. F. 1986. The ecology of deep-sea hydrothermal vent communities. Adv. Mar. Biol. 23:301-362. 23. Haefeli, C., C. Franklin, and K. Hardy. 1984. Plasmid-determined silver resistance in Pseudomonas stutzeri isolated from a silver mine. J. Bacteriol. 158:389-392. 24. Hedges, R. W., and S. Baumberg. 1973. Resistance to arsenic compounds conferred by a plasmid transmissible between strains of Escherichia coli. J. Bacteriol. 115:459-460. 25. Jannasch, H. W., and C. 0. Wirsen. 1973. Deep-sea microorganisms: in situ response to nutrient enrichment. Science 180: 641-643. 26. Jannasch, H. W., and C. 0. Wirsen. 1979. Chemosynthetic primary production at East Pacific Rise sea floor spreading centers. Bioscience 29:592-598. 27. Jannasch, H. W., and C. 0. Wirsen. 1981. Morphological survey of microbial mats near deep-sea hydrothermal vents. Appl. Environ. Microbiol. 41:528-538. 28. Jannasch, H. W., C. 0. Wirsen, S. J. Molyneaux and T. A. Langworthy. 1988. Extremely thermophilic fermentative archaebacteria of the genus Desulforococcus from deep-sea hydrothermal vents. Appl. Environ. Microbiol. 54:1203-1209. 29. Kohn, J. 1953. A preliminary report of a new gelatin liquefaction method. J. Clin. Pathol. 6:249. 30. Kovacs, N. 1956. Identification of Pseudomonas pyocyanae by the oxidase reaction. Nature (London) 178:703. 31. Krieg, N. R., and J. G. Holt. 1984. Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 32. Kupersztoch-Portnoy, Y. M. 1981. Antibiotic resistance of Gram-negative bacteria in Mexico: relationship to drug consumption, p. 529-537. In S. B. Levy, R. C. Clowes, and E. L. Koenig (ed.), Molecular biology, pathogenicity, and ecology of bacterial plasmids. Plenum Publishing Corp., New York. 33. Lanyi, B. 1987. Classical and rapid identification methods for medically important bacteria, p. 1-67. In R. R. Colwell and R. Grigorova (ed.), Methods in microbiology, vol. 19. Academic Press, Inc., New York. 34. Mergeay, M., D. Nies, H. G. Schlegel, J. Gerits, P. Charles, and Van Gisjsegem. 1985. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J. Bacteriol. 162:328-334. 35. Oescher, R., and R. Schmaljohann. 1988. Association of various types of epibacteria with Halicryptus spinulosus (Priapulida). Mar. Ecol. Prog. Ser. 48:285-293. 36. Oppenheimer, C. E., and C. E. Zobell. 1952. The growth and viability of sixty-three species of marine bacteria as influenced by hydrostatic pressure. J. Mar. Res. 11:10-18. 37. Pettibone, M. H. 1984. A new-scale worm commensal with deep-sea mussels on the Galapagos hydrothermal vent (Polychaete: Polynoidae). Proc. Biol. Soc. Wash. 97:226-239. 38. Prieur, D., S. Chamroux, S. Corre, P. Fera, E. Jacq, and G.

APPL. ENVIRON. MICROBIOL.

39.

40. 41.

42.

43. 44.

45.

46. 47.

48.

49.

50. 51.

52. 53.

Mevel. 1989. Quelques caracteristiques des communautes bacteriennes intervenant dans la degradation de la matiere organique en milieu marin, p. 813-827. Atti del Terzo Congresso Nazionale della Societa Italiana di Ecologia, 21-24 ottobre 1987, Siena, Italy. Prieur, D., and C. Jeanthon. 1987. Preliminary study of heterotrophic bacteria isolated from two deep-sea hydrothermal vent invertebrates: Alvinella pompejana (Polychaete) and Bathymodiolus thermophilus (Bivalve). Symbiosis 4:87-98. Ramamoorthy, S., and D. J. Kushner. 1975. Binding of mercuric and other heavy metals ions by microbial growth media. Microb. Ecol. 2:162-176. Rodina, A. G. 1972. Classical and rapid identification methods for medically important bacteria, p. 1-67. In R. R. Colwell and M. S. Zambruski (ed.), Methods in aquatic microbiology. University Park Press, Baltimore. Roesijadi, G., J. S. Young, E. A. Crecelius, and L. E. Thomas. 1985. Distribution of trace metals in the hydrothermal vent clam, Calyptogena magnifica. Biol. Soc. Wash. Bull. 6:311324. Sieburth, J. M. 1975. Microbial seascapes. University Park Press, Baltimore. Sierra, G. 1957. A simple method for the detection of lipolytic activity of microorganisms and some observations on the influence of the contact between cells and fatty substrates. Antonie van Leeuwenhoek J. Microbiol. 23:15-22. Sneath, P. H., and R. R. Sokal. 1973. Numerical taxonomy. W. H. Freeman & Co., San Francisco. Sochard, M. R., D. F. Wilson, B. Austin, and R. R. Colwell. 1979. Bacteria associated with the surface and gut of marine copepods. Appl. Environ. Microbiol. 37:750-759. Sokal, D. M., and C. D. Michener. 1958. A statistical method for evaluating systematic relationship. Univ. Kansas. Sci. Bull. 38:1409-1438. Starr, M. P., H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel. 1981. The prokaryotes, vol. 1 and 2. Springer Verlag KG, Berlin. Straube, W. L., M. O'Brien, K. Davis, and R. Colwell. 1990. Enzymatic profiles of 11 barophilic bacteria under in situ conditions: evidence for pressure modulation of phenotype. Appl. Environ. Microbiol. 56:812-814. Trevors, J. T., K. M. Oddie, and B. H. Belliveau. 1985. Metal resistance in bacteria. Microbiol. Rev. 32:39-54. Tuttle, J. H. 1985. The role of sulfur-oxidizing bacteria at deep-sea hydrothermal vents. Biol. Soc. Wash. Bull. 6:335-343. Tuttle, J. H., C. 0. Wirsen, and H. W. Jannasch. 1983. Microbial activities in the emitted hydrothermal waters of the Galapagos rift vents. Mar. Biol. 73:293-299. Wortman, A. T., and R. R. Colwell. 1988. Frequency and characteristics of plasmids in bacteria isolated from deep-sea amphipods. Appl. Environ. Microbiol. 54:1284-1288.